CN111647551B - Method for extracting and preserving cells from same placenta villus lobule tissue - Google Patents
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Abstract
The invention discloses a method for extracting cells from the same placenta villus lobule tissue, which comprises the following steps: (1) Taking a placenta, and cutting down villus lobule structure tissues in a small block form; (2) Placing the stem cells in a tissue infiltration solution to infiltrate and separate the stem cells remained in the leaflet structure; (3) Selecting a tissue block containing white main blood vessels, stripping or shearing off white velvet Mao Xiaoshe main blood vessel components, adding the tissue block into a buffer solution, and incubating and digesting to obtain the stem cells. The invention also discloses a preservation method: and (3) resuspending the extracted stem cells by using dextran 40, adding the cryopreservation solution, and subpackaging and freezing. The stem cells obtained by the extraction method have high mononuclear cell proportion, high CD34+ or CD133+ cell proportion and high purity. The preservation method of the invention preserves the cells prepared from non-membrane tissues of the same placenta source in a special form to meet the application requirement of precise medical individuation, so that the whole placenta resource is fully utilized, and the preservation method is suitable for the requirement of the future individual use of the cells.
Description
Technical Field
The invention relates to a method for extracting and individually storing cells from the same placenta villus lobule tissue, belonging to the technical field of biomedical engineering.
Background
The placenta, which is an important organ for connecting a fetus and a mother, has a complicated structure and can be classified into tissues containing different types of cells, such as a placenta umbilical cord connection, a amniotic membrane coating, a chorion plate, a villous tissue, a basal decidua and the like. It has been shown that at least different types of cells including hematopoietic (progenitor) stem cells, chorionic Mao Xieguan endothelial stem cells, chorionic, amniotic and chorionic tissue-derived mesenchymal stem cells can be obtained from the same placenta. The placenta is also an important hematopoietic organ in the early development stage of fetus, and researches show that endothelial stem cells and hematopoietic progenitor cells derived from blood vessel components in villus and villus lobule structures of the villus play an important role in the hematopoietic process. Hematopoietic endothelial tissues exist in the early development stage of placenta, mainly in villous lobular structures, vascular endothelium in the placental lobular structures can be shed from vascular endothelium under certain conditions, or hematopoietic endothelial cells lose expression of cadherin 5 and develop into suspension cells in blood (Anderson H et al; blood.2015Dec 24 (26): 2811-2820.), becoming stem cells with hematopoietic differentiation function. After delivery of the fetus, the placenta is not only rich in hematopoietic precursors or hematopoietic (progenitor) stem cells, but also a large number of primitive immune cells can be separated and induced into adoptive immunotherapy cells such as NK by in vitro expansion for clinical adjuvant reinfusion therapy (Kang L et; front Immunol.2013May 1. The number of hematopoietic (progenitor) stem cells (CD 34 +) in cord blood hematopoietic stem cell transplantation is one of the key factors of clinical transplantation, however, the number of single cord blood CD34+ cells is limited, which results in longer transplantation period, higher clinical treatment cost and increased transplantation clinical risk.
The method solves the problem of the number of the current umbilical cord blood hematopoietic stem cells, can improve the implantation efficiency by giving auxiliary lymphocyte infusion after transplantation, and can also reduce GvHD adverse reaction by giving auxiliary mesenchymal stem cell infusion during transplantation. However, these measures generally only employ allogenic lymphocytes or mesenchymal stem cells to assist transplantation, and the introduction of allogenic cells or antigens increases the intensity and probability of negative reactions, thereby increasing the risk of clinical transplantation failure.
The Chinese patent CN 101395266A discloses a method for separating a stem cell group from placenta and preparing an injection solution of a placenta stem cell composition, but the placenta stem cell group is characterized by CD200+ and HLA-G +; CD73+, CD105+, and CD200+; CD200+ and OCT-4+; CD73+, CD105+, and HLA-G +; CD73+ and CD105+; CD34-, CD38-, CD45-, CD73+ and CD105+ show that hematopoietic progenitor cells and stem cells are not included, and the hematopoietic progenitor cells and stem cells separated from the placenta are not involved, so that the waste of important stem cell resources of the placenta is caused; in addition, the patent only establishes a mixed preservation method of the placenta stem cell group, but the characteristics of different cells of tissue parts of the placenta from which stem cells are derived are different, and the optimal conditions required by corresponding cell preservation are also different, so that the mixed preservation of the stem cells can cause the loss of certain types of cells; meanwhile, the method for the ex-warehouse application of the cells in the cell bank according to different individualized requirements is not involved; the existing research shows that the placenta is used as an organ, and cells of different tissue parts have different differentiation directions and the effects of repairing different homologous organs, so that the invention is difficult to meet the personalized treatment requirement realized by the current accurate medical requirement when being applied. Chinese invention patent CN 1407088A discloses a method for separating and preparing hematopoietic stem cells from placenta tissue after collecting umbilical cord blood, the content of the hematopoietic stem cells separated by the technology is small (CD 34+ accounts for 0.3% -0.7%), a large amount of useful active ingredients are not extracted due to the defect of the separation method, and the waste of resources is caused; the technology adopts the traditional well-known cryopreservation method, has great damage to cells, does not meet the requirement of personalized application for bag/tube separation and multiple use and storage according to the precise medical requirement, and is not beneficial to the requirement of benefiting the society after the cell bank is established.
Disclosure of Invention
Aiming at the prior art, aiming at solving the problems that useful resources of placenta tissues are wasted, residual hematopoietic stem cells in the placenta are not fully obtained, the existing placenta stem cells have single preservation mode and large damage to cells, and cannot meet the requirements of repeated use and multi-mode use of precise medical personalized clinical application and the like, the invention establishes an extraction method, a preservation method and an application method of a plurality of cells for assisting umbilical cord blood hematopoietic stem cell transplantation, which provide the same individual placenta source for an autograft patient, and comprises the steps of providing hematopoietic endothelial cells with the function of assisting hematopoietic recovery, so that the efficiency of umbilical cord blood hematopoietic stem cell transplantation is greatly improved, and the risk of transplantation failure is reduced.
The invention is realized by the following technical scheme:
a method for extracting cells from the same placental villus lobule tissue comprises the following steps:
(1) Clipping villus lobule structural tissue: cleaning and disinfecting a placenta, and cutting down villus lobule structure tissues in a small block form, wherein each villus lobule tissue at least comprises 1 thicker main blood vessel for accommodating red colloidal tissue, and the small block of the tissue is red or deep red;
furthermore, the volume of each villus lobule structure tissue is 1-27 cm 3 Generally, 15-20 small pieces of villus lobule structure tissue can be cut from one placenta;
further, the specific way of cutting down the villus lobule structure tissue is as follows: dissecting placenta from son surface, cutting umbilical cord, tearing off chorion plate with surgical forceps to expose main blood vessel of villus lobule, and cutting villus lobule structure tissue with the main blood vessel as center;
the placenta is collected by the following method: selecting and collecting a placenta which is not collected with umbilical cord blood and is provided with an umbilical cord of not less than 10cm, immediately ligating the umbilical cord on the placenta after the placenta is delivered, and extracting hematopoietic stem cells within 24 hours after the placenta is delivered;
furthermore, in order to ensure that the sample is delivered and extracted within 24 hours after delivery, a biomedical sample sterile bag (such as a biomedical sample sterile collection, transportation and preservation combined bag disclosed and protected in Chinese patent CN 107320332A) filled with a buffer solution containing a bacteriostatic agent and a constant-temperature transport box can be adopted to transport the placenta sample to a laboratory at the temperature of 4-10 ℃;
the cleaning is to soak the placenta into normal saline for cleaning; the disinfection is to drip wash 75% medical alcohol on the surface of the placenta tissue for disinfection;
(2) Extracting for the first time: immediately placing the cut small pieces of the villus lobule structure tissue in a tissue infiltration solution,shearing the villus lobule structure tissue in a tissue infiltration solution, and soaking for 15-30 min to infiltrate and separate stem cells remained in the lobule structure; filtering to obtain cell suspension, filtering with 100-150 um cell sieve, centrifuging at 2000rpm for 10min to obtain stem cells, and extracting to obtain 1 × 10 9 ～3×10 9 The proportion of the nucleated cells, CD34+ or CD133+ cells is 3-5%; the placenta tissue with the fully extracted retained cells can be transferred to a new clean centrifuge tube for secondary extraction of stem cells;
the tissue infiltration solution consists of a blood cell protective agent and 0.9 percent of physiological saline solution, wherein the blood cell protective agent accounts for 30 to 90 percent (volume percentage) (the volume ratio of 90 percent, 80 percent, 50 percent and 30 percent can be selected according to the color of villus lobule tissues from deep to light); the tissue infiltration liquid can obviously improve the stem cell separation effect; the cytoprotective agent can be commercially available products such as: the trade name of the blood preservation solution I,
(3) And (3) second extraction: extracting placenta villus lobular tissue blocks with remained cells in the step (2), selecting tissue blocks containing white main blood vessels, peeling off the white main blood vessel components of the villus lobules by using forceps or scissors, and further shearing the remained red tissue into pieces (which are sheared into 1-3 cm) 3 The small blocks) are added into a Du's Phosphate Buffer Solution (DPBS) containing 50-100 ug/mL collagenase, and the mixture is placed into a constant temperature shaking table with the temperature of 37 ℃ and the rpm of 150 for incubation for 30-90 minutes for digestion; after incubation and digestion, filtering to obtain digestive juice, and performing primary incubation and digestion on the residual tissues; mixing the two digestive juices, filtering with 100-150 um cell sieve, centrifuging at 2000rpm for 10min to obtain stem cells, wherein each placenta can obtain 300-800 mL digestive juice, and the extraction can obtain 3 × 10 9 ～9×10 9 The proportion of CD34+ or CD133+ cells is 3-5 percent; the placenta tissue used for digestion in the step is villus lobule tissue which is selected and removed with white villus Mao Xiaoshe as main blood vessel components, and the obtained stem cells are purer than the stem cells obtained by the prior method: is singleThe proportion of the nuclear-like cells is higher (more than 80 percent), and the proportion of the stem cells related to the hemopoiesis (including CD34+ hemopoietic stem cells and CD133 hemopoietic endothelial stem cells) is higher and more than 50 percent.
Furthermore, the volume ratio of the placenta tissue to the Du's phosphate buffer solution is 1:2-3.
The cells separated in the two steps contain different cell components, wherein the cells separated in the first step are cord blood components remaining in placental villus lobules, are more suitable for direct transplantation of combined cord blood, and are separately stored in bags to be used for assisting direct transfusion of hematopoietic stem cell transplantation. In specific application, the two extraction steps (steps 2 and 3) can be carried out in one step according to the needs of customers, and the cells extracted in the first step can be adopted to obtain 0.5 × 10 cells in general 7 ～1×10 7 Cells/g placental villus lobular tissue can be used for assisting in direct transfusion of homologous umbilical cord blood hematopoietic stem cell transplantation, and should be stored in bags separately; specifically, for the sample with more serious placenta damage, the method of the second step can be used for extracting placenta cells, and 3 × 10 placenta cells can be obtained 7 ～5×10 7 Cells/g placental villus lobular tissue.
Further, the method comprises the following steps (a personalized preservation method of the cells derived from the same placental villus lobule tissue):
(4) Re-suspending the stem cells extracted in the steps (2) and (3) by adopting a small amount of dextran 40, adding a freezing solution, subpackaging the mixture into freezing containers, freezing, and carrying out combined transplantation by directly returning or returning after amplification in cooperation with a hematopoietic stem cell transplantation therapy; (ii) a When subpackaging, the stem cells extracted in the steps (2) and (3) can not be mixed; the other cells isolated by the method of the present invention (which means cells other than the stem cells isolated and extracted in step 2) can be used for adoptive immunotherapy, combined lymphocyte infusion-assisted transplantation, combined transplantation with partial hematopoietic stem cell expansion, or the like, as required.
Further, the frozen stock solution is added in two steps: adding a freezing medium A to the cell suspension obtained by resuspending the dextran 40 to resuspend the cells, and then adding a freezing medium B; the frozen stock solution A is a mixed solution of 20% human serum albumin solution (weight volume percent, g/ml) and 15% dextran solution (weight volume percent, g/ml) or 5% glucose injection (weight volume percent, g/ml), and the volume ratio of the two is 3:1; the frozen stock solution B is a mixed solution of a Du's phosphate buffer solution, a 5% glucose solution or a 0.45% sodium chloride injection and dimethyl sulfoxide (DMSO), and the volume ratio of the three is 25; the volume ratio of the frozen stock solution A to the frozen stock solution B is as follows: 1-3:7-9 (namely, A frozen stock solution accounts for 10% -30% of the volume of the total frozen stock solution, B frozen stock solution accounts for 70% -90% of the volume of the total frozen stock solution).
Further, the frozen stock solution is precooled before use, so that the temperature of the frozen stock solution is kept between 2 and 4 ℃ when the frozen stock solution is mixed with cells, and the frozen stock solution is added in a 4 ℃ temperature control table.
Furthermore, the frozen stock solution adopts GMP or pharmaceutical grade solution, and can meet the transfusion requirement after clinical direct resuscitation.
Further, the container can be a freezing bag or a freezing storage tube. The freezing bag can be selected from the cell freezing bag disclosed and protected in Chinese invention patent CN 106577636A, wherein the triple bag can meet the clinical individual requirements of auxiliary feedback after homologous immune cell induced amplification and auxiliary feedback after homologous hematopoietic stem cell amplification, and can meet the adoptive immune cell auxiliary feedback treatment of immune cells for at least 2 times after transplantation. Or 4-5 freezing bags can be adopted, and the freezing volume of each bag can be adjusted between (5-25) mL according to the needs so as to meet the requirement of multiple and irregular placenta cells suitable for homologous transplantation in clinical practice.
Further, the number specification of the dispensed cells is generally 2X 10 7 ～5×10 7 The volume specification is generally 0.5mL, 1mL, 5mL, 10mL, 25mL, 45mL; generally used for assisting hematopoietic stem cell transplantation, one part of placenta-derived cells should be preserved by 1mL, 5mL, 25mL and 45mL, wherein 1mL and 5mL are used for assisting return after induction and amplification of homologous immune cells, and 25mL are used for assisting return after amplification of homologous hematopoietic stem cells. In specific application, the preservation specification of the cells is set according to actual requirements when the maintenance service is provided for the placenta supplier, and generally, the preservation specification can be setThe clinical personalized application scheme for adoptive immunotherapy, combined lymphocyte infusion assisted transplantation, partial hematopoietic stem cell amplification combined transplantation, single or combined use and the like comprises the following specifications: 0.5mL, 1mL, 5mL, 10mL aliquots or single aliquots, each of which may be cryopreserved for use in combination with lymphocyte infusion-assisted transplantation specifications, include: multiple or single portions of 10mL, 15mL, 20mL, 25mL and 35mL, and the freezing specification for partial cell expansion combined transplantation comprises: 10mL, 15mL, 20mL, 25mL, 35mL are combined with each other to give 1 size 1 part by 2 parts or 1 size 2 part by 3 parts.
Further, the cooling method adopted during freezing is generally selected from the following program-controlled cooling methods: keeping at 4 deg.C for 6min; cooling to-10 deg.C at 3 deg.C/min, and maintaining for 15min; reducing the temperature to-50 ℃ at a speed of 1 ℃/min, and keeping the temperature for 7min; the temperature is reduced to-90 ℃ at 40 ℃/min. Reference may also be made to the methods in the Chinese patent application CN 109122665A, CN 108812642A. And after the freezing storage is finished, quickly taking out the frozen product, transferring the frozen product into a freezing storage box, and storing the frozen product in liquid nitrogen.
The placenta stem cell extracted by the method is used for preparing stem cell products; the stem cell product is any one or more than two of stem cells for assisting in returning after the expansion of the homologous hematopoietic stem cells, stem cells for adoptive immunotherapy, stem cells for assisting in transplanting by combined lymphocyte infusion and stem cells for combined transplantation by partial cell expansion.
The placenta stem cells prepared by the method have high hematopoietic stem cell content, and when the cells are stored in multiple bags and multiple tubes, the clinical personalized application schemes such as adoptive immunotherapy, combined lymphocyte infusion assisted transplantation, partial cell amplification combined transplantation and the like can be selected when the cells are conveniently stored in multiple bags and multiple tubes by the method for the client application. The placenta stem cell separation method provided by the invention can obtain the stem cells which are purer than the previous method (the tissue used for extracting the stem cells in the prior art contains tissue components related to non-hematopoiesis, such as white main vascular tissue, so that the separated cells are mixed, the freezing storage is not facilitated, and the personalized selection is not facilitated during the application): the proportion of mononuclear-like cells extracted by the first separation is higher (more than 80 percent), the proportion of hematopoietic-related cells extracted by the second separation (including CD34+ hematopoietic stem cells and CD133 hematopoietic endothelial stem cells) is higher, and the proportion of hematopoietic endothelial stem cells is more than 50 percent.
All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art. To the extent that the terms and phrases are not inconsistent with known meanings, the meaning of the present invention will prevail.
Drawings
FIG. 1: the stem cell classification analysis schematic diagram of the second extraction and separation step shows that the stem cells extracted for the second time by the two separation methods are distributed and concentrated at the position of the mononuclear cells, the proportion accounts for about 90%, and the uniformity is good.
FIG. 2: the flow detection result of the stem cells extracted and separated in the first step before preservation is schematically shown, and the hematopoietic stem cells and the hematopoietic endothelial stem cells respectively account for 2.72 percent and 2.63 percent.
FIG. 3: the flow detection result before preservation of the stem cells extracted and separated in the second step is shown schematically, wherein the hematopoietic stem cells account for 18.71 percent, and the hematopoietic endothelial stem cells account for 54.19 percent.
FIG. 4 is a schematic view of: the apoptosis detection result after cell cryopreservation is shown in a schematic diagram, wherein C2 and C3 expression Annexin-FITC represents the proportion of apoptotic cells, the lower left quadrant represents normal cells, and the proportion of normal cells is 96.6% and 99.3% respectively.
Detailed Description
The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purposes of this invention, the invention is nevertheless described herein in as detail as possible.
The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.
Example 1
After consent of hospitals and lying-in women is obtained, selecting and collecting a placenta which is not collected with umbilical cord blood and is provided with umbilical cord of not less than 10cm, immediately ligating the umbilical cord at the upper end of the placenta by a professional after the placenta is delivered, transferring the sample into a sterile biological sample bag filled with buffer solution containing bacteriostatic agents, sealing the sample bag, putting the sample bag into a constant-temperature transport box, and taking heat preservation measures to ensure that the sample is transported at the temperature of 4-10 ℃ and delivered to a laboratory for cell extraction within 24 hours. After the test, the sample and transportation conformance check is carried out, and the transportation maximum temperature is confirmed to be lower than 10 ℃, and the transportation time is within 20h, as shown in table 1.
TABLE 1
| Sample number | Transport time (h) | Maximum sample temperature | Sample weight (g) | Leaflet tissue weight (g) |
| 201901S | 15 | 10℃ | 480.0 | 137.0 |
| |
10 | 5℃ | 265.4 | 100.5 |
| 201903S | 12 | 10℃ | 367.0 | 158.8 |
| 201904S | 8 | 6℃ | 281.7 | 129.3 |
| 201905S | 18 | 8℃ | 450.0 | 158.8 |
| Average | 12.6 | 7.8 | 368.82 | 136.88 |
The received qualified placenta samples were placed on a GMP laboratory clean bench and the placenta was washed 3 times, 4 times, 5 times and sterilized 2 times, 3 times, 4 times on both sides and the collateral tissue, respectively, each time according to a volume (mL) ratio of 75% medical alcohol followed by 0.9% normal saline followed by cleaning and sterilizing according to 200. When the amnion on the surface of the son-surface placenta is damaged or has obvious stain, the target part can be washed by 50-200mL 75% medical alcohol and 100-300mL 0.9% physiological saline outside a clean workbench, and the condition that no pollution is introduced when a sample is dissected is ensured.
Example 2
The cleaned placenta sample is taken and placed on a sterile tray, the placenta is dissected from the son surface, the umbilical cord is firstly cut off from the root of the placenta, a chorion plate is torn off from the remained incision by a pair of surgical forceps to expose the major vessels of the villus lobules, the structural tissues containing the villus lobules are cut off by taking the major vessels of the major stems as the center, 15mL of cytoprotective agent (trade name blood preservation solution I,
) In a 50mL centrifuge tube. 15-20 tissue blocks containing the villus and lobular structures of major trunks and great vessels can be cut from the same placenta, and the volume of each tissue block is 1-27 cm 3 . The weight of the Mao Xiaoshe tissue in each tube is generally 20-30 g, the tissue is cut into pieces in buffer solution by surgical scissors, the cut tissue is fully stirred and extruded to separate the cells remained in the leaflet structure, and the placenta tissue after cell separation is transferred to a new clean centrifuge tube to be subjected to secondary stem cell extraction.
Filtering the cell suspension with 150 μm cell sieve, centrifuging the cell filtrate at 2000rpm for 10min to obtain 1 × 10 cells per placenta 9 ～3×10 9 The proportion of the nucleated cells, CD34+ or CD133+ cells is 3-5%. The extracted cells are resuspended by 1mL of dextran 40 after centrifugation, and then a proper amount of cryopreservation protective agent is added to ensure that the final concentration of the cells is 2-5 multiplied by 10 7 The combined type cell freezing bag is divided into two freezing storage tubes of 0.5mL and 1mL according to the actual needs of customers, or 5mL + (20-25) mL or (10-15) mL + (35-45) mL are filled into combined type cell freezing bags with corresponding specifications, wherein the cells stored in the freezing storage tubes of 0.5mL and 1mL and the combined type cell freezing bag of 5mL can be used for adoptive immunotherapy, the combined type cell freezing bags of 20-25 mL and (35-45) mL can be used for combined lymphocyte infusion assisted transplantation, and the combined type cell freezing bags of 5mL and 10mL can be used for assisted transplantation after hematopoietic stem cells are expanded. The specifications for the different batches of samples are shown in table 2.
TABLE 2
Example 3
Taking villus lobule tissue after shearing and cell buffer solution infiltration and primary cell separation, transferring the villus lobule tissue into 4-6 new centrifuge tubes in a superclean bench, further shearing, weighing to cut the tissue into 1-3 cm 3 Adding DPBS and collagenase into the tissue according to the volume ratio of the tissue to the DPBS of 1:3, wherein the concentration of the enzyme is 80 mug/mL, and incubating the digestion solution in a constant-temperature shaking table with the temperature of 37 ℃ and the rotating speed of 150rpm for 60min and 90min. And filtering out the residual tissues in the digestion solution, and performing incubation digestion for 20-30 min under the same conditions again according to the method. Finally, 300-800 mL of digestive juice can be obtained from each part of placenta, and 3X 10 can be obtained 9 ～9×10 9 The proportion of the nucleated cells, CD34+ or CD133+ cells is 3-5%. Filtering the extracted cell suspension with a 150-micron cell sieve, centrifuging the cell filtrate at 2000rpm for 10min, resuspending the cell precipitate with 1mL of dextran 40, and adding a proper amount of cryopreservation protective agent to make the final concentration of the cells be 2-5 × 10 7 /mL，The cells are respectively packed into two freezing tubes of 0.5mL and 1mL, or 5mL + (20-25) mL or (10-15) mL + (35-45) mL according to the actual needs of a client and are packed into a combined cell freezing bag with a corresponding specification, wherein the cells stored in the freezing tubes of 0.5mL and 1mL and the combined cell freezing bag of 5mL can be used for adoptive immunotherapy, the combined cell freezing bag of 20-25) mL and (35-45) mL can be used for assisted transplantation by combining lymphocyte infusion, and the freezing bags of 5mL and 10mL can be used for assisted transplantation after hematopoietic stem cells are expanded. As shown in fig. 1 and table 3.
TABLE 3
Example 4
The cells extracted by shearing or digestion separation are taken, two cells are taken from each sample, and 10uL of each of PE, PE-Cy7-IgG/FITC-CD45 and FITC-CD45/PE-CD34/PE-Cy7-CD133 is added for flow cytometry detection, and the results are shown in fig. 2 and fig. 3.
Example 5
When the cells extracted in examples 2 and 3 were stored, 1mL of dextran 40 was added to the cell pellet and resuspended, and the cell count was performed, and the cell concentration was adjusted to 2 to 5X 10 according to the storage volume specification 7 mL, calculate the total final cell preservation volume. Adding solution A (accounting for 20% of the total volume of the frozen stock solution) into the cell suspension, placing in a refrigerator at 4 ℃ for 15min, and adding solution B (accounting for 80% of the total volume of the frozen stock solution), wherein the solution A is 20% of human serum albumin: mixed solution with volume ratio of 15% dextran/5% glucose injection =3:1, and solution B is DPBS:5% of glucose: DMSO volume ratio = 25. The method for cooling during freezing can refer to the method in Chinese patent application CN 109122665A. The components of the frozen stock solution in the method are GMP-grade solutions.
The frozen stock solutions A and B are pre-cooled before use, so that the temperature of the frozen stock solutions A and B is kept between 2 and 4 ℃ when the frozen stock solutions A and B are mixed with cells, and the frozen stock solutions A and B are added in a temperature control table at 4 ℃.
The program-controlled cooling method comprises the following steps: keeping the temperature for 6min at 2-8 ℃; cooling to-10 deg.C at 3 deg.C/min, and maintaining for 15min; reducing the temperature to-50 ℃ at a speed of 1 ℃/min, and keeping the temperature for 7min; freezing at 40 deg.C/min to-90 deg.C, quickly taking out, transferring to freezing box, and storing in liquid nitrogen.
Example 6
Taking cells which are frozen for not less than 7 days, unfreezing the cells in a water bath at the temperature of 35-38 ℃, taking two cells from each sample, respectively detecting the cell viability and the apoptosis rate, adding 10uL of PI/FITC-annexin7 into an apoptosis detection tube, and analyzing by flow cytometry, wherein the result is shown in figure 4.
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims. All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each such publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.
Claims (4)
1. A method for extracting cells from the same placental villus lobule tissue is characterized by comprising the following steps:
(1) Clipping villus lobule structural tissue: cleaning and disinfecting a placenta, and cutting down villus lobule structure tissues in a small block form, wherein each villus lobule tissue at least comprises 1 thicker main blood vessel for accommodating red colloidal tissue, and the small block of the tissue is red or deep red;
the specific mode of shearing down the villus lobule structural tissue is as follows: dissecting placenta from son surface, cutting umbilical cord, tearing off chorion plate with surgical forceps to expose main blood vessel of villus lobule, and cutting villus lobule structure tissue with the main blood vessel as center;
(2) Extracting for the first time: immediately placing the cut villus lobular structure tissue small blocks into a tissue infiltration liquid, cutting the villus lobular structure tissue into pieces in the tissue infiltration liquid, and soaking for 15-30 min to ensure that stem cells remained in the lobular structure are infiltrated and separated; filtering to obtain cell suspension, filtering with a cell sieve, and centrifuging to obtain stem cells;
(3) And (3) second extraction: extracting the placental villus lobular tissue blocks with the retained cells in the step (2), selecting the tissue blocks containing white main blood vessels, stripping or shearing off the white villus Mao Xiaoshe main blood vessel components, further shearing the left red tissues, adding the red tissues into a buffer solution, wherein the buffer solution is a Duchen phosphate buffer solution containing collagenase, and incubating and digesting; after incubation and digestion, filtering to obtain digestive juice, and performing primary incubation and digestion on the residual tissues; mixing the digestive juice of the two times, filtering by a cell sieve, and centrifuging to obtain stem cells;
(4) And (4) re-suspending the stem cells extracted in the steps (2) and (3) by using dextran 40 respectively, adding the freezing solution, and subpackaging the mixture into a freezing container for freezing.
2. The method of claim 1, wherein the cells derived from the same placental villus lobule tissue are extracted by: the volume of each villus lobule structure tissue is 1-27 cm 3 Cutting 15-20 small pieces of villus lobule structure tissue from one placenta;
the placenta is collected by the following method: selecting and collecting placenta which is not collected with umbilical cord blood and is left with umbilical cord no less than 10cm, ligating umbilical cord on placenta immediately after delivery of placenta, and extracting hematopoietic stem cells within 24 hours after delivery;
the cleaning is to soak the placenta into normal saline for cleaning; the disinfection is to drip wash 75% medical alcohol on the surface of the placenta tissue for disinfection;
the tissue infiltration solution consists of a blood cell protective agent and a normal saline solution, wherein the blood cell protective agent accounts for 30-90%.
3. The method for extracting cells derived from the tissue of the same placental villus leaflet as claimed in claim 1, wherein the cryopreservation solution is added in two steps: adding a freezing solution A to the cell suspension re-suspended by the dextran 40 to re-suspend the cells, and then adding a freezing solution B;
the frozen stock solution A is a mixed solution of 20% human serum albumin solution and 15% dextran solution or 5% glucose injection, and the volume ratio of the frozen stock solution A to the dextran solution is 3:1;
the frozen stock solution B is a mixed solution of a Du's phosphate buffer solution, a 5% glucose solution or a 0.45% sodium chloride injection and dimethyl sulfoxide, and the volume ratio of the three is 25;
the volume ratio of the frozen stock solution A to the frozen stock solution B is as follows: 1 to 3:7 to 9.
4. The method of claim 1, wherein the cells derived from the same placental villus lobule tissue are extracted by: the freezing container is selected from a 3-linked cell freezing bag, a 4-linked cell freezing bag or a 5-linked cell freezing bag, and the number specification of the split-packaged cells is 2 multiplied by 10 7 ～5×10 7 The volume specification is selected from 0.5mL, 1mL, 5mL, 10mL, 25mL and 45mL.
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| CN103789262A (en) * | 2014-02-17 | 2014-05-14 | 宁波普莱森特生物科技有限公司 | Preparation method and preservation method of clinical application-level placental hematopoietic stem cells |
| CN105368780A (en) * | 2015-12-14 | 2016-03-02 | 广州赛莱拉干细胞科技股份有限公司 | Lavage fluid, enzymatic hydrolysate and method for separating placenta hematopoietic stem cells |
| CN108148806A (en) * | 2018-02-02 | 2018-06-12 | 中国人民解放军军事科学院军事医学研究院 | A kind of fast separating process of placental hematopoietic stem cell |
| CN108812642A (en) * | 2018-07-18 | 2018-11-16 | 银丰生物工程集团有限公司 | A kind of systems approach and application for preparing placenta tissue according to layer of structure and freezing |
| CN111254113A (en) * | 2018-12-03 | 2020-06-09 | 北京银丰鼎诚生物工程技术有限公司 | Method for extracting placenta hematopoietic stem cells |
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| EP1442115B9 (en) * | 2001-11-15 | 2009-12-16 | Children's Medical Center Corporation | Methods of isolation, expansion and differentiation of fetal stem cells from chorionic villus, amniotic fluid, and placenta and therapeutic uses thereof |
| US8574899B2 (en) * | 2010-12-22 | 2013-11-05 | Vladimir B Serikov | Methods for augmentation collection of placental hematopoietic stem cells and uses thereof |
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| CN103789262A (en) * | 2014-02-17 | 2014-05-14 | 宁波普莱森特生物科技有限公司 | Preparation method and preservation method of clinical application-level placental hematopoietic stem cells |
| CN105368780A (en) * | 2015-12-14 | 2016-03-02 | 广州赛莱拉干细胞科技股份有限公司 | Lavage fluid, enzymatic hydrolysate and method for separating placenta hematopoietic stem cells |
| CN108148806A (en) * | 2018-02-02 | 2018-06-12 | 中国人民解放军军事科学院军事医学研究院 | A kind of fast separating process of placental hematopoietic stem cell |
| CN108812642A (en) * | 2018-07-18 | 2018-11-16 | 银丰生物工程集团有限公司 | A kind of systems approach and application for preparing placenta tissue according to layer of structure and freezing |
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